Abstract Breakthroughs in deep-sequencing and gnotobiotic animal model systems have established that the gut microbiota, the trillions of bacteria that live within the gastrointestinal tract, play important roles in normal mammalian physiology and transitions to disease. However, the exact reactions catalyzed by microbial enzymes and their influence on mammalian tissues still remain poorly understood. The study of microbial enzymes is challenging because the gut microbiome encodes roughly five million proteins ? not a proteome that can be tackled easily. As outlined here, we have focused on a specific set of gut microbial enzymes that play critical roles in reversing mammalian metabolic processes that are crucial in responses to a range of therapeutic agents. These microbial enzymes did not co-evolve with their mammalian hosts to process drugs; instead, they naturally act on the abundant inactivated metabolites of hormones and neurotransmitters that reach the gut. In this proposal, we concentrate on two hormones, the primary circulating thyroid hormone thyroxine and the cancer-promoting steroid estrone, and one neurotransmitter, serotonin, that are all processed by Phase II drug metabolizing UDP-glucuronosyltransferase enzymes that attach inactivating glucuronic acid sugar moieties to mark these compounds for excretion. Thyroxine, estrone, and serotonin metabolites reach the gut as glucuronide conjugates and are subject to reactivation by the focus of our project ? the intestinal microbiome-encoded b-glucuronidase (GUS) enzymes that cleave off the glucuronic acid sugar. We have pioneered the study of gut microbial GUS enzymes and have established the roles they play in drug efficacy and toxicity, and have developed GUS-targeted inhibitors that improve the treatment of disease in animal models. We have also shown that there are 279 unique GUS orthologs in the human gut microbiome. Here, our overarching hypotheses are that the human gut microbiome encodes a range of structurally ? and functionally ? distinct GUS enzymes capable of acting on chemically discrete, endobiotic glucuronide substrates and that such enzymes are susceptible to selective inhibition by novel chemotypes or existing drugs. We will test these hypotheses by completing three aims focused on the endobiotic-glucuronide conjugates of thyroid hormones, estrogens, and neurotransmitters. The results we obtain will crucially advance our basic understanding of the chemical crosstalk between human tissues and the microbiota and may lead to novel approaches for the treatment of hormone imbalances, cancer, gut health, cardiovascular disease, and even psychological disorders.